The major goal of this project is to develop functional probes to measure extracellular pH in breast tumors models using Cerenkov imaging, a novel optical imaging technique. Low pH in the tumor microenvironment (TME) results from the high levels of lactic acid produced from increased glycolysis inside the cell and subsequent transport to the extracellular space. This low pH is a hallmark of many solid tumors and has been linked to invasion and increased tumor aggressiveness. Several enzymes have been identified as contributing to the decrease in pH including monocarboxylate transporter, sodium/proton antiporter and carbonic anhydrase. One of the most important enzymes associated with pH decrease in the extracellular matrix is lactate dehydrogenase, catalyzing the intracellular conversion of pyruvate into lactate. Cerenkov radiation is a phenomenon arising from decaying radionuclides. This phenomenon was discovered in the 1930s but its application to imaging was not identified until late-2000s; with translation to humans occurring for the first time in 2013. Cerenkov radiation occurs when subatomic particles, such as electrons and positrons, are emitted from a decaying radionuclide and travel faster than the speed of light in their medium. Breaking this speed limit causes the production of multispectral photons along the path traveled by the particle. The presence of a pH-sensitive chromophore, such as a pH indicator, will absorb Cerenkov photons at and near the absorption maxima of the dye. This creates a disruption in the Cerenkov emission spectrum and the attenuation in the signal can be measured and quantified ratiometrically or by correlation to an external standard curve. Cerenkov radiation will allow us to image pH-sensitive molecules that are not fluorescent, which will open the door for more bioactive molecules to be developed for in vivo imaging. In this application we will test the hypothesis that Cerenkov imaging can detect changes in absorption of a functional chromophore in response to changes in pH in the TME. The hypothesis will be tested by the following Specific Aims: Specific Aim 1: Synthesis and Characterization of pH-dependent 18F-labeled chromophores that functionally absorb Cerenkov radiation: This will include identification of suitable pH indicators, synthesis of fluorinated derivatives, chemical and optical characterization of the products and studies for cell toxicity. Addition of 18F fluorine atoms to te molecule will provide a Cerenkov photon source that will be modulated by pH changes. Specific Aim 2: Application of Cerenkov-absorbing contrast agents in mouse models: The application of the chromophores in tumor and non-tumor bearing mice will determine biodistribution and optimal delivery of the probe via tail vein into the tumors. Orthotopic tumors, using 4175 (highly metastatic variant of MDA-MB-231 cells) and LDH knockdown cells, will be used to most accurately depict the breast cancer TME.